Over the past several years, we have been studying two phenomena in cloned populations of CD4 positive T lymphocytes referred to as costimulation and anergy. Both affect the production of the T cell growth factor interleukin-2 (IL-2) produced by these cells. Costimulation entails a 30 to 100-fold enhancement of IL-2 production when signaling through the antigen-specific T cell receptor is supplemented with signaling through the CD28 receptor on the same cell. Anergy is an anti-proliferative state that the T cell enters when it only receives a signal through the antigen-specific receptor. In this case, subsequent stimulation of IL-2 production is inhibited 10-50-fold. Our goals are to try and understand the molecular mechanisms behind these two phenomena and to explore their relevance in vivo.Recently, we have set up a new model for studying tolerance to persistent low dose antigen in vivo, which results in the generation of a large number of anergic T cells. We inject CD4+, cytochrome c-specific T cells from a T cell receptor transgenic mouse on a Rag2-/- background (a monospecific T cell population) into a second transgenic mouse expressing the cytochrome c antigen under the control of the MHC class I promoter and an immunoglobin heavy chain enhancer. Within 24 hours after transfer, the T cells are all activated by the antigen (as evidenced by an increase in size and expression of CD69), and proliferate extensively for several days, increasing in number about 100-fold. This expansion is followed by a deletional phase during which 50% of the cells disappear. Finally, the population reaches a steady state level in which the cells appear to be refractory to restimulation in vivo and in vitro. In the anergic state, IL-2 responses to high doses of antigen in vitro are inhibited 90%. The T cells also make significant amounts of interferon (IFN) gamma at high antigen doses, but little IL-4, IL-6 or IL-10. Expression of early activation markers, such as CD69 and CD25, on restimulation is greatly impaired, and initial biochemical studies of signal transduction show a 2 fold inhibition of Early Response Kinase (ERK) phosphorylation following T cell receptor engagement. This hyporesponsive state is reversible if the cells are transferred again into a second host not expressing the antigen. Interestingly, if the retransfer is into a host expressing antigen, the cells initially show an expansion phase, although the rate is 3 fold less than for a naive cell. Within one week, however, the population has again become hyporesponsive and in fact IFN gamma production under optimal stimulation conditions is 10 fold lower than before, suggesting the possibility of an even deeper state of anergy.